Dendritic cells (DCs) are known in the art. In absence of lineage specific markers, they are generally identified by the lack of leukocyte markers of other lineages (CD3 for T cell lineage, CD14 and CD15 for monocytic and granulocyte lineages, CD19, CD20 and CD24 for B cell lineage and CD16, CD56 and CD57 for NK cell lineage) by their specific immunophenotype (positive for surface antigen CD40, CD80, CD86), and their morphology (characterized by the presence of dendrites or membrane processes) (1-3).
From the functional point of view, DCs are known to be highly potent antigen-presenting cells (APC), playing in vivo a pivotal role in the priming of the immune response (1-3). In this connection, a main distinction is made between mature and immature DCs.
Immature DCs are weak initiators of immune response specialized in capturing and processing antigens, phenotypically characterized by low expression of the accessory molecules CD40, CD80, CD86 and the lack of CD83 expression. Upon appropriate stimuli, DCs undergo extensive changes: loss of antigen-capturing function and the upregulation of the expression of costimulatory molecules (CD40, CD80 and CD86) together with the induction of CD83 and CD25 (1-4).
Terminally differentiated/mature DCs are instead capable of readily priming naive T cells within lymphoid tissues.
Phenotype of DCs in the mature state is characterized accordingly by the production of a variety of cytokines, including typically IL-15, (1-3, 5, 6) which are considered capable to affect, by autocrine/paracrine mechanisms, the phenotype and functional activity of DCs themselves as well as of other host cells (7-9).
Phenotype of mature/activated DCs is also characterized by specific chemotactic properties. In this connection, it is well known in the art that migration of DCs is tightly regulated as a function of maturation (10-13).
Thus, immature DCs respond to inflammatory chemokines, such as MIP-1α, MIP-1β, RANTES and MIP-3α (14) as a consequence of the expression of the chemokine receptors CCR5 and CCR6, while mature DCs have lost their responsiveness to most of these chemokines, as a result of down-regulation of cognate receptor expression or activity (15).
Conversely, mature DCs have been reported to respond to MIP-3β/ELC and 6Ckine/SLC as a consequence of the induction of their specific receptor CCR7 which is lacking on immature DCs (10, 11, 15).
On the other hand, DCs are themselves producer of a series of chemokines. Upon maturation, DCs have an initial burst of Mip-3α, Mip-3β and IL-8, whereas RANTES and MCP-1 are produced in a more sustained fashion. The production of MIP-3β/ELC by activated/mature DCs is also important in supporting the generation of the immune response by recruiting naive T and B cells, which selectively express CCR7.
Mature DCs express also IP-10, a potent chemoattractant for activated/memory Th1 cells by binding to the receptor CXCR3 (10, 16, 17), while immature DCs express MDC and TARC attracting specifically chronically activated Th2 lymphocytes. (10, 18). In addition, in presence of mature DCs and IL-12, T-helper cells turn into IFNγ-producing Th1 cells, which promote the cellular arm of the immune response, whereas CD8+ cytotoxic T cells are induced to proliferate vigorously. IFNγ and IL-12 promote further the differentiation of T cells into killer cells.
Accordingly, mature DCs are considered capable of stimulating the outgrowth and activation of a variety of T cells.
The ability to prime antigen-specific naive T cells represent a unique and critical function of DCs. Moreover, by virtue of their enhanced expression of HLA and costimulatory molecules, DCs stimulate allogeneic MLR (which allows comparison of the capacity of different APCs to stimulate T cell proliferation independently of the antigen) more efficiently than any other antigen presenting cell. Thus, there is a growing interest in utilizing such cells as cellular adjuvants for prophylactic or therapeutic vaccination toward infectious agents or tumors.
However, the use of DCs has been limited by their very low frequency in peripheral blood and the invasiveness of procedures aimed to gain access to bone marrow or lymphoid organs. Such limitations render complicate and expensive obtaining DCs to be used as adjuvant and application related thereto.
Consequently, some processes allowing production of DCs in vitro have been defined. These procedures are all based on the information that DCs originate from progenitor CD34+ cells in bone marrow and blood or can be derived from peripheral blood mononuclear CD14+ cells (19, 20). Hence, according to a first approach DCs are generated by cultivation of CD34+ progenitors in medium containing Flt3-L or SCF (stem cell factor), followed by a combinations of various cytokines including GM-CSF, IL-4, and TNFα (3, 4).
In a second approach, an initial phase of cultivation of progenitors CD34+ cells is carried out in the presence of GM-CSF, TNF-α and IL-4 (PCT/AU97/00801) followed by treatment with type I IFN.
Following a further approach, CD34+ precursor cells from cord blood or bone marrow are cultivated in presence of IL-3 or GM-CSF (21). Thus, this procedure has been shown to induce cell proliferation, which is strongly potentiated by TNFα and culminates in the appearance of CD1a+ cells displaying typical DC morphology and surface markers. CD34+ precursor cells cultured in the presence of GM-CSF and TNFα differentiate into two distinct DC populations within 5-7 days, as defined by the exclusive expression of CD14 and CD1a. However, by further culturing, CD1a expression is generally downregulated just as CD83 appears (3).
According to a fourth approach, immature DCs are generated starting from peripheral blood CD14+ monocytes cultivated in GM-CSF in conjunction with IL-13 or IL-4 for 5-7 days. DCs produced according to this procedure, however, display features of and behave as immature DCs expressing low levels of CD80 and CD86. Consequently, these DCs act as weak stimulators of a specific T cell response and MLR. In this setting, further DC maturation can be driven by the addition of TNFα, IL-1, LPS, monocyte-conditioned medium (22) or sCD40L for two additional days (2, 3).
Thus, the requirement of a further step for DC maturation by addition of other factors to immature DCs represents a strong limitation for the rapid generation of DCs highly effective for clinical purposes. Moreover, it is not clear whether the use of mature DCs represents an advantage over immature DCs for clinical applications. In this context, DCs endowed with intermediate phenotypic and functional properties, i.e.: high phagocytic activity associated to the expression of membrane markers typical of mature DCs and to a potent immunostimulatory capacity, would represent a novel cellular entity of great interest for clinical applications.